EP1613759A1 - Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients - Google Patents
Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrientsInfo
- Publication number
- EP1613759A1 EP1613759A1 EP04725049A EP04725049A EP1613759A1 EP 1613759 A1 EP1613759 A1 EP 1613759A1 EP 04725049 A EP04725049 A EP 04725049A EP 04725049 A EP04725049 A EP 04725049A EP 1613759 A1 EP1613759 A1 EP 1613759A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nitrogen
- carbon
- medium
- fermentation
- process according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/626—Natamycin; Pimaricin; Tennecetin
Definitions
- the present invention relates to the field of fermentative production of desired compounds, such as secondary metabolites, proteins or peptides.
- the actinomycetes a family of filamentous bacteria, are of great importance for the fermentation industry. Many members of this family are known to produce secondary metabolites or extracellular enzymes and several of these products of bacterial metabolism have an industrial application.
- the bacteria are generally cultivated in liquid media (submerged cultures), leading to excretion of the products into the liquid, from which they can be isolated. Formation of product can take place during the initial fast growth of the organism and/or during a second period in which the culture is maintained in a slow-growing or non-growing state.
- the amount of product which is formed per unit of time during such a process is generally a function of a number of factors: the intrinsic metabolic activity of the organism; the physiological conditions prevailing in the culture (e.g.
- the viscosity of a culture fluid is determined by a number of factors such as the composition of the medium, the presence and nature of products excreted by the microorganisms, and (most important) the morphology of the microorganism. If one could influence the morphological characteristics of the microorganisms in a positive way (i.e. to decrease the specific viscosity), the process could be operated at a higher production rate or a higher concentration of bacteria could be achieved. Both changes in the process would result in a higher productivity.
- the present invention provides a fermentation process for the production of a desired compound comprising culturing a filamentous bacterial strain in a liquid fermentation medium, wherein the carbon containing nutrients and nitrogen containing nutrients are maintained at low concentrations in the fermentation medium.
- a feed comprising carbon and nitrogen containing nutrients is supplied to the medium and the nutirients in the feed are in such a ratio that low concentrations of both carbon and nitrogen containing nutrients are maintained in the culture.
- the filamentous bacteria are preferably of the family Actinomyces, more preferably of the genus Streptomyces.
- Bacterial strains of the family Actinomycetes are known to produce desired compounds, which have commercial applications, such as secondary metabolites, proteins and peptides. Examples thereof are natamycin, nistatine, glucose isomerase and clavulanic acid.
- the actinomycetes strains Streptomyces natalensis and Streptomyces silvosporens produce the antifungal compound natamycin, which has several applications as an antifungal compound.
- Fermentation processes comprising such filamentous bacteria are generally characterised by two phases. Usually the process starts with a phase where growth of the microorganism occurs until conditions for growth become unfavourable, for instance because one of the growth supporting nutrients becomes depleted from the medium. The initial (batch) phase may be followed by a phase where the microorganisms are maintained in a viable state. Often most of the product of interest is formed in this second phase.
- more nutrients may be supplied to the culture, either discontinuously as a single or repeated charge of fresh nutrients, or continuously by feeding one or more nutrients containing fluids in to the fermentation vessel.
- This mode of fermentation is called fed-batch fermentation.
- a fermentation process may be further prolonged by removing part of the fermentation mash, for instance when the fermentation vessel becomes completely filled as a result of feeding with nutrient containing fluids. This process form is called extended fermentation or repeated (fed-)batch fermentation.
- the initial (batch) phase will end when one of the nutrients is depleted. This phase may be followed by measuring the oxygen uptake which will decrease towards the end of the initial phase. In general, the initial phase will take 6 to 48 hours.
- the second phase starts when feeding of the nutrients is started. Feeding of nutrients allows the continuation of the fermentation process for a longer period than is possible in simple batch fermentation process.
- the optimal ratio of carbon and nitrogen containing nutrients can be determined by the skilled person, depending on the elementary composition of the organism and the product(s), the effect of the N/C ratio on the physiology of the organism and, more specifically, the product forming capacity of the organism. It has been found that neither carbon excess nor nitrogen excess will lead to the desired result. In the optimal situation, both the available carbon and nitrogen will be almost depleted from the medium at the end of the batch process and/or during the process of prolonged fed-batch type fermentation.
- the concentration of the nitrogen containing nutrient in the medium during the second phase is preferably less than 0.5 g/l, more preferably less than 0.25 g/l and most preferably less than 0.1 g/l (expressed as gram of nitrogen per litre).
- the concentration of the carbon containing nutrient is preferably less than 5 g/l, more preferably less than 2.5 g/l and most preferably less than 1 g/l (expressed as gram of carbon per litre).
- the feed can be supplied as one feed containing all the nutrients or preferably as more than one subfeeds each comprising either a nitrogen containing nutrient, a carbon containing nutrient or a combination of nitrogen and carbon containing nutrients.
- the feed is also controlled in such a way that the amount of oxygen is between 20 and 70% of air saturation, preferably between 30 and 60% of air saturation.
- Oxygen typically in the form of air, is generally introduced at or near the bottom of the fermentor.
- One of more nozzles are installed for the introduction of air or another oxygen containing gas such as (purified) oxygen.
- a stirrer is present in the reactor to stimulate the oxygen uptake. Moreover, the stirrer prevents concentration gradients of the feed or subfeed developing in the fermentor.
- Figure 1 Viscosity development of a nitrogen excess-culture (•) and a nitrogen-carbon double-limited culture ( ⁇ ).
- Figure 2 Agitation power required to control the dissolved oxygen concentration at a 30% air saturation. Both cultures, nitrogen excess (o) and nitrogen-carbon double-limited (o), were operated under otherwise similar process conditions.
- Figure 3 Viscosity development of a nitrogen excess culture (o) and a nitrogen-carbon double-limited culture ( ⁇ ).
- Figure 4 Product accumulation in a nitrogen excess culture ( Q ) and a nitrogen-carbon double-limited culture (#).
- Figure 5 Full scale fermentation of Streptomyces natalensis to produce natamycin.
- the initial process (•) used a limiting feed of soybean oil, while the NH3 concentration was kept at a non-limiting level.
- the improved process ( ⁇ ) the NH3 concentration was kept at a low value by continuous feeding of a NH3 solution in proportion to the oil feeding rate.
- the reduced culture viscosity allowed faster feeding of oil.
- the increase in product formation was approximately proportional to the increase in oil feeding rate.
- Steptomyces natalensis strain ATCC27448 was cultivated in 2000ml conical shake containing 500 mL growth medium of the following composition: g/L
- the pH was adjusted to 7.0 by adding NaOH/H 2 S0 4 , and the medium was sterilized by autoclavation (20 minutes at 120°C).
- the content of a full-grown shake flask was used to inoculate a fermentation vessel containing 6L medium of the following composition: g/L
- composition of the trace element solution was as follows: g/L Citric acid.1H 2 0 175
- the temperature and pH of the medium were controlled at 25°C and 7.0 respectively. Dissolved oxygen concentration was kept above 30% of air saturation, by increasing airflow and/or stirrer speed when necessary.
- the culture entered the second phase of fermentation.
- a second feeding line was installed to feed ammonia.
- the average feeding rate of the soybean oil was 3 g/h.
- Ammonia was supplied in proportion to the soybean oil feeding rate.
- a series of fermentations were carried out, in which different ammonia feeding rates were applied while keeping the soybean oil feeding rate constant. For this strain, the carbon source and the nitrogen source were totally consumed when the ratio of NH3 to oil was in the range of 30-40 mg NH3/g oil.
- Figure 1 Under nitrogen excess conditions the viscosity reaches the usual high values. Under conditions of simultaneous carbon and nitrogen limitation, the viscosity drops to a much lower value, causing better aeration conditions. For a good production it is preferred that the dissolved oxygen concentration is maintained at a level of above 30% of air saturation.
- Figure 2 illustrates that for maintaining this dissolved oxygen concentration much less agitation power (energy) is needed when the culture is under a condition of nitrogen-carbon double limitation.
- Example 2 Another fermentation experiment was carried out using the same procedure as described in Example 1 using a strain of Streptomyces natalensis. This strain is a producer of the anti-fungal compound natamycin. In this experiment two fermentations were run. One experiment was under carbon limitation and nitrogen excess (NH 3 level was kept at 150-200 mg/L during the oil feeding phase). The second experiment was run under nitrogen-carbon double limitation during the oil feeding phase, employing a NH 3 /oil ratio of 32 mg/g. Some results are shown in Figure 3 and 4. It is obvious that a very significant difference in viscosity was observed between the two modes of fermentation. A low viscosity is very beneficial for efficient process operation. However, a low viscosity coupled with a poor product formation potency would be negative. In this experiment, the product formation was not affected at all by the conditions leading to low viscosity (Figure 3). The rate of product formation in the nitrogen-carbon double limitation experiment is faster in the second part of the fermentation despite a slightly slower start.
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10178179A EP2287324A3 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
EP04725049A EP1613759B1 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03100896 | 2003-04-04 | ||
PCT/EP2004/003662 WO2004087934A1 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
EP04725049A EP1613759B1 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10178179.7 Division-Into | 2010-09-22 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1613759A1 true EP1613759A1 (en) | 2006-01-11 |
EP1613759B1 EP1613759B1 (en) | 2012-08-01 |
Family
ID=33104167
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10178179A Withdrawn EP2287324A3 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
EP04725049A Expired - Lifetime EP1613759B1 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10178179A Withdrawn EP2287324A3 (en) | 2003-04-04 | 2004-04-01 | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients |
Country Status (9)
Country | Link |
---|---|
US (1) | US20060270004A1 (en) |
EP (2) | EP2287324A3 (en) |
JP (1) | JP2006521801A (en) |
CN (1) | CN1768146B (en) |
BR (1) | BRPI0409074A (en) |
CA (1) | CA2521419A1 (en) |
DK (1) | DK1613759T3 (en) |
MX (1) | MXPA05010639A (en) |
WO (1) | WO2004087934A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9410175B2 (en) | 2005-03-24 | 2016-08-09 | Dsm Ip Assets B.V. | Process for microbial production of a valuable compound |
ES2694810T3 (en) | 2013-05-31 | 2018-12-27 | Dsm Ip Assets B.V. | Microbial agriculture |
CN105431045A (en) * | 2013-05-31 | 2016-03-23 | 帝斯曼知识产权资产管理有限公司 | Microbial agriculture |
CN114875100B (en) * | 2022-06-27 | 2023-06-16 | 山东第一医科大学(山东省医学科学院) | Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3892850A (en) * | 1956-03-13 | 1975-07-01 | Gist Brocades Nv | Pimaricin and process of producing same |
US4480034A (en) * | 1982-06-10 | 1984-10-30 | Celanese Corporation | Continuous fermentation process and bioconversion-product recovery |
US5182207A (en) * | 1984-09-14 | 1993-01-26 | American Cyanamid Company | Strains of streptomyces thermoarchaensis |
NZ215788A (en) | 1985-04-12 | 1988-04-29 | Weston George Ltd | Continuous ethanol production by bacterial fermentation |
US5902579A (en) | 1991-08-05 | 1999-05-11 | Bio-Technical Resources | Natamycin-containing streptomyces biomass and its use in animal feed |
EP0796916A1 (en) | 1996-03-22 | 1997-09-24 | Triple-A B.V. | Improvement of amino acid fermentation processes |
-
2004
- 2004-04-01 CN CN2004800092116A patent/CN1768146B/en not_active Expired - Fee Related
- 2004-04-01 WO PCT/EP2004/003662 patent/WO2004087934A1/en active Application Filing
- 2004-04-01 CA CA002521419A patent/CA2521419A1/en not_active Abandoned
- 2004-04-01 EP EP10178179A patent/EP2287324A3/en not_active Withdrawn
- 2004-04-01 US US10/551,178 patent/US20060270004A1/en not_active Abandoned
- 2004-04-01 DK DK04725049.3T patent/DK1613759T3/en active
- 2004-04-01 BR BRPI0409074-8A patent/BRPI0409074A/en not_active Application Discontinuation
- 2004-04-01 EP EP04725049A patent/EP1613759B1/en not_active Expired - Lifetime
- 2004-04-01 JP JP2006505021A patent/JP2006521801A/en not_active Withdrawn
- 2004-04-01 MX MXPA05010639A patent/MXPA05010639A/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO2004087934A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP2287324A2 (en) | 2011-02-23 |
EP2287324A3 (en) | 2011-06-29 |
CN1768146A (en) | 2006-05-03 |
EP1613759B1 (en) | 2012-08-01 |
WO2004087934A1 (en) | 2004-10-14 |
JP2006521801A (en) | 2006-09-28 |
BRPI0409074A (en) | 2006-03-28 |
MXPA05010639A (en) | 2005-12-15 |
DK1613759T3 (en) | 2012-10-29 |
US20060270004A1 (en) | 2006-11-30 |
CN1768146B (en) | 2010-05-26 |
CA2521419A1 (en) | 2004-10-14 |
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